To sharpen the focus on the study of neurotransmitter receptor regulation, we have concentrated our efforts on the serotonin receptor system and discontinued the work on muscarinic receptors. We have reported that in cerebellar granule cells (CGC), persistent stimulation of 5-HT2A receptors with a receptor agonist leads to paradoxical increase in the level of 5-HT2A receptor binding sites and mRNA. This agonist up-regulation requires de novo RNA and protein synthesis and requires receptor-mediated calcium entry and a calmodulin-dependent event such as calcium/calmodulin kinase activation. We have further characterized this agonist-up-regulated process in CGC. and have confirmed the increase in 5-HT2A receptor protein after agonist stimulation by immunoblotting using a monoclonal antibody specific for 5-HT2A receptors. Immunocytochemical studies using the same antibody revealed that 5-HT2A receptors are located on both neurites and cell bodies of CGC. In contrast to an increase in 5-HT2A receptor protein, the levels of Gq/11 protein (-subunits were found to be unchanged after agonist treatment. Unexpectedly, the levels of Gs protein (-subunits are significantly increased after protracted agonist stimulation, possibly the result of receptor cross-talk. Electrophoretic mobility shift assays show that transcription factor binding to the consensus sequences of AP-1 and CRE is markedly enhanced by agonist stimulation in a time-dependent manner. The increased AP-1 binding is blocked by a 5-HT2A receptor antagonist, ritanserin, and markedly suppressed by inhibitors of calcium/calmodulin-dependent kinases. Supershift assays revealed that CREB and Jun D are common components of both AP-1 and CRE binding complexes. Moreover, we found that 5-HT2A receptor stimulation also increases the level of p-CREB protein. The binding of p-CREB transcription factor to the AP-1 site suggests that CREB may modulate the transcription of genes that contain AP-1, but lack CRE sites in their promoters, through interaction with this AP-1 site. The rat 5-HT2A receptor up-regulation may involve such a mechanism. We also found that several 5-HT2A receptor antagonists such as mianserin, spiperone, ketanserin, and methylsergide possess -intrinsic activity+ capable of up-regulating 5-HT2A receptors in CGC. This intrinsic activity could be related to the antidepressant effects of certain 5-HT2A receptor antagonists, especially mianserin. Unlike the effects of agonists, mianserin-induced 5-HT2A receptor up-regulation does not involve a change in AP-1 DNA binding activity. Moreover, c-Fos mRNA is unaffected by agonist treatment, but is rapidly and transiently down-regulated by mianserin, whose actions are similar to those of other antidepressants. Thus, our results demonstrate that distinct mechanisms underlie homologous up-regulation of 5-HT2A receptor sites elicited by agonist and antagonist, suggesting an involvement of different signaling pathways for these two processes.